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1.
Reprod Toxicol ; 110: 150-160, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35460820

RESUMO

Amphetamine derivatives negatively impact serotonin (5-HT) production, which triggers apoptosis in different tissues, depending on the receptor they bind. 5-HT in the ovary stimulates estradiol secretion, a survival factor of granulosa cells. The effect of amphetamine derivatives on the serotonergic system of the ovary and follicular development is unknown. Therefore, in this study, we investigated the effects of p-chloroamphetamine (pCA), derived from amphetamines, on estradiol production, follicular development, apoptosis of granulosa cells, and serotonin 5-HT7 receptor (R5-HT7) expression. Female rats (30 days old) were injected with 10 mg/kg of pCA intraperitoneally and were euthanized 48 or 120 h after treatment. The concentration of 5-HT in the hypothalamus decreased at 48 and 120 h after treatment and in the ovary at 120 h. The serum concentration of estradiol decreased at all times studied. Follicular atresia, TUNEL-positive (apoptotic) granulosa cells and Bax expression were elevated by pCA, but none of these effects was associated with R5-HT7 expression. These results suggest that pCA induces the dysregulation of the serotonergic system in the hypothalamus and the ovary, negatively impacting estradiol production and follicular development.


Assuntos
Atresia Folicular , Serotonina , Anfetamina , Animais , Apoptose , Estradiol/metabolismo , Feminino , Atresia Folicular/fisiologia , Células da Granulosa/metabolismo , Ratos , p-Cloroanfetamina/farmacologia
2.
Food Chem Toxicol ; 141: 111395, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32437895

RESUMO

p-Chloroamphetamine (PCA), an amphetamine derivative, has been shown to induce serotonergic toxicity. However, the precise mechanism of serotonergic toxicity induced by PCA remains unclear. In this study, PCA treatment (20 mg/kg, i.p.) did not significantly change 5-HT1A receptor gene expression, but significantly increased 5-HT2A receptor gene expression. Furthermore, 5-HT2A receptor antagonist MDL11939, but not 5-HT1A receptor antagonist WAY100635, significantly attenuated PCA-induced serotonergic impairments. We investigated whether PCA activated a specific isoform of protein kinase C (PKC), since previous evidence indicated the involvement of PKC in neurotoxicity induced by amphetamines. We observed that PCA treatment significantly increased the expression levels of PKCδ among all PKC isoforms. MDL11939 treatment significantly attenuated PCA-induced phosphorylation of PKCδ. However, PCA-induced increase in 5-HT2A receptor gene expression was not altered by rottlerin (a pharmacological inhibitor of PKCδ) in mice, suggesting that 5-HT2A receptor is an upstream molecule for the activation of PKCδ. Rottlerin or PKCδ knockout significantly attenuated serotonergic behaviors. However, MDL11939 did not show any additional effects against the attenuation caused by PKCδ knockout in mice, suggesting that PKCδ gene is a molecular target for 5-HT2A receptor-mediated serotonergic effects. Our results suggest that 5-HT2A receptor mediates PCA-induced serotonergic impairments via activation of PKC.δ.


Assuntos
Proteína Quinase C-delta/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Serotoninérgicos/farmacologia , p-Cloroanfetamina/farmacologia , Animais , Camundongos , Fosforilação
3.
Neurotox Res ; 37(3): 543-552, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31939043

RESUMO

Serotoninergic nerves are known to modulate sensitization of dopamine receptors (DA-R) in a rodent model of Parkinson's disease (PD). However, serotoninergic nerves are not known to have a prominent role on DA exocytosis in intact rats. The current study was undertaken to explore the possible influence of serotoninergic nerves on DA exocytosis in Parkinsonian rats. Rat pups were treated at 3 days after birth with the neurotoxin 6-hydroxydopamine (6-OHDA; 134 µg icv, half into each lateral ventricle; desipramine, 1 h pretreatment), in order to produce marked long-lasting destruction of neostriatal dopaminergic innervation, as evidenced by the 90-95% depletion of DA (p < 0.001) [HPLC/ED] into adulthood. Controls received vehicle/desipramine in place of 6-OHDA. Other groups received the serotoninergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 25 µg base, icv, half in each lateral ventricle; desipramine, 1 h; 75 mg/kg pargyline HCl, 30 min) at 3 days post-birth; or both 6-OHDA+5,7-DHT treatments. In adulthood, an in vivo microdialysis study was undertaken to ascertain that p-chloroamphetamine (PCA, 1 mM in the microdialysate)-evoked DA release in the neostriatum was reduced approximately 50% in the 6-OHDA group, while PCA-evoked DA release in the 6-OHDA+5,7-DHT group was substantially increased, to a level equivalent to that of the vehicle control. The baseline neostriatal microdialysate level of 3,4-dihydroxyphenylacetic acid (DOPAC) was also higher in the 6-OHDA+5,7-DHT group vs 6-OHDA group; also, during the 2nd hour of PCA infusion. PCA-enhanced DA exocytosis occurred in the absence of changes in hydroxyl radical (HO·) in the microdialysate (i.e., assay of 2,3- and 2,5-dihydroxybenzoic acid, 2,3-DHBA; 2,5-DHBA). The overall findings demonstrate that an adulthood serotoninergic nerve lesion enhanced PCA-evoked DA exocytosis in a rodent model of severe PD, while susceptibility to oxidative stress was unchanged. The implication is that serotoninergic nerves may normally suppress the release of DA and/or act as an uptake site and storage sink for accumulated DA in parkinsonian-like neostriatum. Potentially, serotoninergic agonists or antagonists, targeting subtype-selective serotonin receptors, may be viable therapeutic adjuncts in PD.


Assuntos
5,7-Di-Hidroxitriptamina/toxicidade , Dopamina/metabolismo , Exocitose/efeitos dos fármacos , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Oxidopamina/toxicidade , Doença de Parkinson/metabolismo , p-Cloroanfetamina/administração & dosagem , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Masculino , Microdiálise , Ratos , Serotonina/metabolismo
4.
ACS Chem Neurosci ; 10(1): 190-200, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30540906

RESUMO

Although G protein-coupled receptors (GPCRs) are recognized as pivotal drug targets involved in multiple physiological and pathological processes, the majority of GPCRs including orphan GPCRs (oGPCRs) are unexploited. GPR88, a brain-specific oGPCR with particularly robust expression in the striatum, regulates diverse brain and behavioral functions, including cognition, mood, movement control, and reward-based learning, and is thus emerging as a novel drug target for central nervous system disorders including schizophrenia, Parkinson's disease, anxiety, and addiction. Nevertheless, no effective GPR88 synthetic ligands have yet entered into clinical trials, and GPR88 endogenous ligands remain unknown. Despite the recent discovery and early stage study of several GPR88 agonists, such as 2-PCCA, RTI-13951-33, and phenylglycinol derivatives, further research into GPR88 pharmacology, medicinal chemistry, and chemical biology is urgently needed to yield structurally diversified GPR88-specific ligands. Drug-like pharmacological tool function and relevant signaling elucidation will also accelerate the evaluation of this receptor as a viable neurotherapeutic target.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Doenças do Sistema Nervoso/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cromanos/administração & dosagem , Cromanos/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Humanos , Doenças do Sistema Nervoso/tratamento farmacológico , Receptores Acoplados a Proteínas G/genética , p-Cloroanfetamina/administração & dosagem , p-Cloroanfetamina/análogos & derivados , p-Cloroanfetamina/metabolismo
5.
Syst Biol Reprod Med ; 64(5): 340-347, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29788785

RESUMO

Spermatogenesis and steroidogenesis are testicular functions regulated by gonadotrophins as well as other factors, including serotonin. Testicular serotonin acts as an autocrine regulator of testosterone secretion, but studies on its role in spermatogenesis and sperm quality are scarce. Here, we analyzed the effects of intratesticular inhibition of serotonin synthesis on gonadotrophins, testosterone, and sperm quality. Both testicles of 30-day-old rats were injected once with saline solution (SS) or distinct concentrations of p-chloroamphetamine (PCA) (0.03, 0.06, or 0.12 mg). At 65 days of age, rats were euthanized and sperm density, motility, membrane integrity, mitochondrial function, and abnormalities were evaluated in gametes from the vas deferens. Inhibition of synthesis of intratesticular serotonin by PCA diminished the concentrations of testosterone and follicle-stimulating hormone (FSH) but luteinizing hormone (LH) levels were unaltered. Sperm density was not modified in animals injected with the different concentrations of PCA. In contrast, the percentage of sperm with abnormalities increased and the sperm membrane integrity decreased in animals injected at higher PCA concentrations. The functionality of sperm mitochondria in PCA-injected animals decreased only at the highest PCA dose. Our results indicate that testicular serotonin plays a role in testosterone synthesis and in the normal development of sperm, and blocking its effects disrupts the hormonal communication between the testis and hypophysis. ABBREVIATIONS: SS: saline solution; PCA: p-chloroamphetamine; FSH: follicle-stimulating hormone; LH: luteinizing hormone; TPH: tryptophan hydroxylase; MAO: monoamine oxidase; AC: absolute control group; PI: propidium iodide; FLICA: fluorescence inhibitor of caspase; 3ß-HSD: 3ß-hydroxysteroid dehydrogenase; 17-KSR: 17-ketosteroid reductase; DHT: 5-dihydrotestosterone.


Assuntos
Gonadotropinas/metabolismo , Serotonina/fisiologia , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testosterona/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hormônio Foliculoestimulante/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Ratos , Serotonina/biossíntese , Serotoninérgicos/administração & dosagem , Serotoninérgicos/farmacologia , Motilidade Espermática/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/fisiologia , Testículo/fisiologia , p-Cloroanfetamina/administração & dosagem , p-Cloroanfetamina/farmacologia
6.
J Biomed Sci ; 24(1): 23, 2017 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-28347302

RESUMO

BACKGROUND: GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions. METHODS: In this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader. RESULTS: The CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z'-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays. CONCLUSIONS: We have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.


Assuntos
Cálcio/metabolismo , Cromanos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/agonistas , p-Cloroanfetamina/análogos & derivados , Animais , Células CHO , Cricetulus , Relação Estrutura-Atividade , p-Cloroanfetamina/farmacologia
7.
Autophagy ; 12(11): 2197-2212, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27560450

RESUMO

The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.


Assuntos
Arginina/metabolismo , Autofagia , Proteínas/toxicidade , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Proteína Huntingtina/metabolismo , Marcação por Isótopo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Camundongos , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteômica , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , p-Cloroanfetamina/farmacologia , Proteínas tau/metabolismo
8.
Neuron ; 91(4): 748-762, 2016 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-27499084

RESUMO

It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Studies using fixed tissue have suggested that serotonin neurons might be a notable exception, but remain inconclusive. We have employed in vivo two-photon microscopy to produce time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and subsequent slow recovery of axonal density, which is dominated by new growth with little contribution from local sprouting. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and followed by regrowth from cut ends into and across the stab rift zone. Regrowing serotonin axons do not follow the pathways left by degenerated axons. The regrown axons release serotonin and their regrowth is correlated with recovery in behavioral tests.


Assuntos
Axônios/fisiologia , Lesões Encefálicas/patologia , Neocórtex/citologia , Neocórtex/fisiologia , Regeneração Nervosa/fisiologia , Neurônios Serotoninérgicos/fisiologia , Animais , Lesões Encefálicas/fisiopatologia , Camundongos , Camundongos Transgênicos , Neocórtex/patologia , Reflexo de Sobressalto/fisiologia , Degeneração Retrógrada/induzido quimicamente , Neurônios Serotoninérgicos/citologia , Neurônios Serotoninérgicos/patologia , Imagem com Lapso de Tempo , p-Cloroanfetamina/toxicidade
9.
ACS Chem Neurosci ; 7(10): 1418-1432, 2016 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-27499251

RESUMO

GPR88, an orphan receptor richly expressed in the striatum, is implicated in a number of basal ganglia-associated disorders. In order to elucidate the functions of GPR88, an in vivo probe appropriate for CNS investigation is required. We previously reported that 2-PCCA was able to modulate GPR88-mediated cAMP production through a Gαi-coupled pathway. Early structure-activity relationship (SAR) studies suggested that the aniline moiety of 2-PCCA is a suitable site for diverse modifications. Aimed at elucidating structural requirements in this region, we have designed and synthesized a series of analogues bearing a variety of substituents at the phenyl ring of the aniline moiety. Several compounds (e.g., 5j, 5o) showed improved or comparable potency, but have lower lipophilicity than 2-PCCA (clogP 6.19). These compounds provide the basis for further optimization to probe GPR88 in vivo functions. Computational studies confirmed the SAR trends and supported the notion that 4'-substituents on the biphenyl ring exit through a largely hydrophobic binding site to the extracellular loop.


Assuntos
Cromanos/química , Cromanos/farmacologia , Neurotransmissores/química , Neurotransmissores/farmacologia , Receptores Acoplados a Proteínas G/agonistas , p-Cloroanfetamina/análogos & derivados , Sequência de Aminoácidos , Compostos de Anilina/química , Animais , Sítios de Ligação , Células CHO , Cromanos/síntese química , Cricetulus , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Estrutura Molecular , Neurotransmissores/síntese química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-Atividade , p-Cloroanfetamina/síntese química , p-Cloroanfetamina/química , p-Cloroanfetamina/farmacologia
10.
Neurotox Res ; 29(4): 569-82, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26846719

RESUMO

Abused amphetamines, such as d-amphetamine (AMPH) and methamphetamine (METH), are highly addictive and destructive to health and productive lifestyles. The abuse of these drugs is associated with impulsive behavior, which is likely to contribute to addiction. The amphetamines also differentially damage dopamine (DA) and serotonin (5-HT) systems, which regulate impulsive behavior; therefore, exposure to these drugs may differentially alter impulsive behavior to effect the progression of addiction. We examined the impact of neurotoxicity induced by three amphetamines on impulsive action using a stop-signal task in rats. Animals were rewarded with a food pellet after lever pressing (i.e., a go trial), unless an auditory cue was presented and withholding lever press gained reward (i.e., a stop trial). Animals were trained on the task and then exposed to a neurotoxic regimen of either AMPH, p-chloroamphetamine (PCA), or METH. These regimens preferentially reduced DA transporter levels in striatum, 5-HT transporter levels in prefrontal cortex, or both, respectively. Assessment of performance on the stop-signal task beginning 1 week after the treatment revealed that AMPH produced a deficit in go-trial performance, whereas PCA did not alter performance on either trial type. In contrast, METH produced a deficit in stop-trial performance (i.e., impulsive action) but not go-trial performance. These findings suggest that the different neurotoxic consequences of substituted amphetamines are associated with different effects on inhibitory control over behavior. Thus, the course of addiction and maladaptive behavior resulting from exposure to these substances is likely to differ.


Assuntos
Encéfalo/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/toxicidade , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Comportamento Impulsivo/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/fisiopatologia , Animais , Temperatura Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Dextroanfetamina , Modelos Animais de Doenças , Dopamina/metabolismo , Masculino , Metanfetamina , Atividade Motora/efeitos dos fármacos , Testes Neuropsicológicos , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina , Fatores de Tempo , p-Cloroanfetamina
11.
Reprod Fertil Dev ; 28(6): 806-14, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25486044

RESUMO

Male germ-cell apoptosis occurs naturally and can be increased by exposure to drugs and toxic chemicals. Individuals may have different rates of apoptosis and are likely to also exhibit differential sensitivity to outside influences. Previously, we reported that p-chloroamphetamine (pCA), a substance that inhibits serotonin synthesis, induced germ-cell apoptosis in prepubertal male rats. Here, we identified prepubertal rats with naturally high or low rates of germ-cell apoptosis and evaluated gene expression in both groups. Bax and Shbg mRNA levels were higher in rats with high rates of germ-cell apoptosis. Rats were then treated with pCA and the neuro-hormonal response and gene expression were evaluated. Treatment with pCA induced a reduction in serotonin concentrations but levels of sex hormones and gonadotrophins were not changed. Rats with initially high rates of germ-cell apoptosis had even higher rates of germ-cell apoptosis after treatment with pCA. In rats with high rates of germ-cell apoptosis Bax mRNA expression remained high after treatment with pCA. On the basis of category, an inverse relationship between mRNA expression of Bax and Bcl2, Bax and AR and Bax and Hsd3b2 was found. Here we provide evidence that innate levels of germ-cell apoptosis could be explained by the level of mRNA expression of genes involved with apoptosis and spermatogenesis.


Assuntos
Apoptose , Regulação da Expressão Gênica no Desenvolvimento , Serotonina/metabolismo , Maturidade Sexual , Espermatogênese , Espermatogônias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ácido Hidroxi-Indolacético/metabolismo , Cinética , Masculino , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Serotoninérgicos/farmacologia , Maturidade Sexual/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , p-Cloroanfetamina/farmacologia
12.
J Vet Med Sci ; 78(1): 71-6, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26321443

RESUMO

Chemoreceptor cells aggregating in clusters in the chicken thoracic aorta contain 5-hydroxytryptamine (5-HT) and have voltage-dependent ion channels and nicotinic acetylcholine receptors, which are characteristics typically associated with neurons. The aim of the present study was to investigate the effects of 5-HT uptake inhibitors, fluvoxamine, fluoxetine and clomipramine (CLM), and amphetamine derivatives, p-chloroamphetamine (PCA) and methamphetamine (MET), on endogenous 5-HT outflow from the isolated chick thoracic aorta in vitro. 5-HT was measured by using a HPLC system with electrochemical detection. The amphetamine derivatives and 5-HT uptake inhibitors caused concentration-dependent increases in endogenous 5-HT outflow. PCA was about ten times more effective in eliciting 5-HT outflow than MET. The 5-HT uptake inhibitors examined had similar potency for 5-HT outflow. PCA and CLM increased 5-HT outflow in a temperature-dependent manner. The outflow of 5-HT induced by PCA or 5-HT uptake inhibitors was independent of extracellular Ca(2+) concentration. The 5-HT outflow induced by CLM, but not that by PCA, was dependent on the extracellular NaCl concentration. These results suggest that the 5-HT uptake system of 5-HT-containing chemoreceptor cells in the chicken thoracic aorta has characteristics similar to those of 5-HT-containing neurons in the mammalian central nervous system (CNS).


Assuntos
Aorta Torácica/efeitos dos fármacos , Metanfetamina/farmacologia , Inibidores de Captação de Serotonina/farmacologia , Serotonina/metabolismo , p-Cloroanfetamina/farmacologia , Animais , Aorta Torácica/metabolismo , Galinhas , Clomipramina/farmacologia , Fluoxetina/farmacologia , Fluvoxamina/farmacologia , Técnicas In Vitro , Masculino
13.
Pharmacol Rep ; 67(1): 90-6, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25560581

RESUMO

BACKGROUND: we investigated the possible mechanisms involved in the interactions of venlafaxine (VFX), a selective serotonin and noradrenaline reuptake inhibitor, and morphine (MRF), an opioid receptor agonist, after acute and chronic VFX treatment in diabetic neuropathic pain model (DNPM). METHODS: The studies were performed on male rats. The changes in nociceptive thresholds were determined by using mechanical stimuli (the Randall-Selitto and the von Frey tests). Diabetes was induced by intramuscular administration of streptozotocin. In order to investigate the mechanism of interaction, animals were also pretreated with naloxone (NLX), a nonselective opioid antagonist, yohimbine (YOH), a nonselective α2-adrenergic antagonist, and p-chloroamphetamine (PCA), a neurotoxin that destroys serotonergic neurons. The µ-opioid receptors' density was determined with the use of radioligand binding assay. RESULTS: VFX potentiated antinociceptive action of MRF after acute administration of VFX and this effect was decreased by pretreatment of NLX, YOH and PCA. On the contrary, VFX administered for 21 days prior to MRF significantly decreased the analgesic action of MRF; this effect was augmented only after YOH pretreatment. Also, 21-days administration of VFX caused decreasing tendency in the number of µ-opioid receptors in the brain stem. CONCLUSIONS: The results of our study show that single administration of VFX potentiates antinociceptive action of morphine in DNPM. This effect is probably mediated by both, noradrenergic and serotonergic systems. On the other hand, 21-days administration of VFX significantly decreases analgesic action of MRF. Moreover, there is a possibility that VFX acts as an antagonist of N-methyl-d-aspartate receptors.


Assuntos
Analgésicos Opioides/uso terapêutico , Analgésicos/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Morfina/uso terapêutico , Neuralgia/tratamento farmacológico , Cloridrato de Venlafaxina/uso terapêutico , Antagonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Analgésicos Opioides/antagonistas & inibidores , Animais , Tronco Encefálico/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Sinergismo Farmacológico , Masculino , Morfina/antagonistas & inibidores , Limiar da Dor , Estimulação Física , Ratos , Receptores Opioides mu/efeitos dos fármacos , Serotoninérgicos/farmacologia , Cloridrato de Venlafaxina/antagonistas & inibidores , Ioimbina/uso terapêutico , p-Cloroanfetamina/farmacologia
16.
Mol Imaging Biol ; 17(2): 239-47, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25267548

RESUMO

PURPOSE: p-Chloroamphetamine (PCA) is a neurotoxin that selectively degenerates the serotonin (5-HT) axon terminals. In order to study the brain metabolic consequences induced by serotonergic denervation, a single dose of PCA (2.5 or 10 mg/kg i.p.) was administered to male adult rats. PROCEDURES: In vivo regional brain metabolism was evaluated 3 and 21 days after PCA (2.5 or 10 mg/kg; i.p.) injection by 2-deoxy-2-[(18)F] fluoro-D-glucose ([(18)F] FDG) positron emission tomography (PET). At day 22, the following markers of neurotoxicity were determined: (a) 5-HT axon terminal lesion by 5-HT transporter (SERT) autoradiography, (b) reactive gliosis by glial fibrillary acidic protein immunohistochemistry, and (c) eventual neurodegeneration by DAPI/Fluoro-Jade C labeling. RESULTS: An average of 20 % reduction of [(18)F] FDG uptake in most brain areas was observed at day 21 under 10 mg/kg PCA treatment. Instead, 2.5 mg/kg PCA only reduced metabolic activity in neocortex. Likewise, the high dose of PCA exerted a strong decrease (>30 %) in SERT density in several 5-HT innervated regions, but no effect was found in midbrain raphe nuclei, the main source of serotonergic neurons. Although PCA induced astroglial activation both in hippocampus and cortex in response to axotomy, no signs of neuronal death in these areas were detected. CONCLUSIONS: Overall, [(18)F] FDG PET revealed that the reduction of the brain metabolic activity induced by PCA is related to 5-HT axon terminal lesion, with no apparent affectation of neuronal viability.


Assuntos
Fluordesoxiglucose F18 , Hipocampo/diagnóstico por imagem , Hipocampo/metabolismo , Neurotoxinas/toxicidade , Tomografia por Emissão de Pósitrons , p-Cloroanfetamina/toxicidade , Animais , Autorradiografia , Fluoresceínas/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Glucose/metabolismo , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , Indóis/metabolismo , Masculino , Degeneração Neural/diagnóstico por imagem , Degeneração Neural/patologia , Ratos Sprague-Dawley , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo
17.
Sci Rep ; 4: 6344, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25212999

RESUMO

In the arginylation branch of the N-end rule pathway, unacetylated N-terminal destabilizing residues function as essential determinants of protein degradation signals (N-degron). Here, we show that a neurostimulant, para-chloroamphetamine (PCA), specifically inhibits the Arg/N-end rule pathway, delaying the degradation of its artificial and physiological substrates, including regulators of G protein signaling 4 (RGS4), in vitro and in cultured cells. In silico computational analysis indicated that PCA strongly interacts with both UBR box and ClpS box, which bind to type 1 and type 2 N-degrons, respectively. Moreover, intraperitoneal injection of PCA significantly stabilized endogenous RGS4 proteins in the whole mouse brain and, particularly, in the frontal cortex and hippocampus. Consistent with the role of RGS4 in G protein signaling, treatment with PCA impaired the activations of GPCR downstream effectors in N2A cells, phenocopying ATE1-null mutants. In addition, levels of pathological C-terminal fragments of TDP43 bearing N-degrons (Arg208-TDP25) were significantly elevated in the presence of PCA. Thus, our study identifies PCA as a potential tool to understand and modulate various pathological processes regulated by the Arg/N-end rule pathway, including neurodegenerative processes in FTLD-U and ALS.


Assuntos
Arginina/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , p-Cloroanfetamina/farmacologia , Animais , Linhagem Celular Tumoral , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas RGS/metabolismo
18.
ACS Chem Neurosci ; 5(7): 576-87, 2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-24793972

RESUMO

GPR88 is an orphan G-protein-coupled receptor (GPCR) enriched in the striatum. Genetic deletion and gene expression studies have suggested that GPR88 plays an important role in the regulation of striatal functions and is implicated in psychiatric disorders. The signal transduction pathway and receptor functions of GPR88, however, are still largely unknown due to the lack of endogenous and synthetic ligands. In this paper, we report the synthesis of a GPR88 agonist 2-PCCA and its pure diastereomers, which were functionally characterized in both transiently and stably expressing GPR88 HEK293 cells. 2-PCCA inhibited isoproterenol-stimulated cAMP accumulation in a concentration-dependent manner in cells expressing GPR88 but not in the control cells, suggesting that the observed cAMP inhibition is mediated through GPR88 and that GPR88 is coupled to Gαi. 2-PCCA did not induce calcium mobilization in GPR88 cells, indicating no Gαq-mediated response. A structure-activity relationship (SAR) study of 2-PCCA was also conducted to explore the key structural features for GPR88 agonist activity.


Assuntos
Cromanos/síntese química , Cromanos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , p-Cloroanfetamina/análogos & derivados , Cálcio/metabolismo , Cromanos/química , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Isoproterenol/farmacologia , Receptores Acoplados a Proteínas G/genética , Transfecção , p-Cloroanfetamina/síntese química , p-Cloroanfetamina/química , p-Cloroanfetamina/farmacologia
19.
ACS Chem Neurosci ; 5(5): 329-34, 2014 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-24661118

RESUMO

Dopaminergic neurotransmission has been investigated extensively, yet direct optical probing of dopamine has not been possible in live cells. Here we image intracellular dopamine with sub-micrometer three-dimensional resolution by harnessing its intrinsic mid-ultraviolet (UV) autofluorescence. Two-photon excitation with visible light (540 nm) in conjunction with a non-epifluorescent detection scheme is used to circumvent the UV toxicity and the UV transmission problems. The method is established by imaging dopamine in a dopaminergic cell line and in control cells (glia), and is validated by mass spectrometry. We further show that individual dopamine vesicles/vesicular clusters can be imaged in cultured rat brain slices, thereby providing a direct visualization of the intracellular events preceding dopamine release induced by depolarization or amphetamine exposure. Our technique opens up a previously inaccessible mid-ultraviolet spectral regime (excitation ~270 nm, emission < 320 nm) for label-free imaging of native molecules in live tissue.


Assuntos
Encéfalo/citologia , Encéfalo/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Técnicas In Vitro , Espectrometria de Massas , Camundongos , Microscopia Ultravioleta , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Imagem Óptica , Ratos , Serotoninérgicos/farmacologia , Fatores de Tempo , p-Cloroanfetamina/farmacologia
20.
Br J Pharmacol ; 171(4): 1007-18, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24251585

RESUMO

BACKGROUND AND PURPOSE: Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), noradrenaline (NET) and 5-HT (SERT); influx of amphetamine leads to efflux of substrates. Various models have been proposed to account for this amphetamine-induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine-induced conformation that is not likely in alternative access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis. EXPERIMENTAL APPROACH: Xenopus laevis oocytes and HEK293 cells expressing human (h) SERT were voltage-clamped and exposed to 5-HT, p-chloroamphetamine (pCA) or methylenedioxyamphetamine (MDMA). KEY RESULTS: In contrast to the currents induced by 5-HT, pCA-triggered currents through SERT decayed slowly in Xenopus laevis oocytes once the agonist was removed (consistent with the molecular stent hypothesis). However, when SERT was expressed in HEK293 cells, currents induced by 3 or 100 µM pCA decayed 10 or 100 times faster, respectively, after pCA removal. CONCLUSIONS AND IMPLICATIONS: This discrepancy in decay rates is inconsistent with the molecular stent hypothesis. In contrast, a multistate version of the alternative access model accounts for all the observations and reproduces the kinetic parameters extracted from the electrophysiological recordings. A crucial feature that explains the action of amphetamines is their lipophilic nature, which allows for rapid diffusion through the membrane.


Assuntos
Modelos Biológicos , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Serotoninérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/fisiologia , Serotonina/farmacologia , p-Cloroanfetamina/farmacologia , Animais , Células HEK293 , Humanos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Xenopus laevis
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